Seeing Cells in 3D

How Light-Based Tomography is Revolutionizing Tissue Engineering

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The Quest for the Third Dimension

For decades, biologists grew cells in flat, two-dimensional dishes—a convenient but fundamentally flawed approach. Just as studying fish in puddles reveals little about ocean life, 2D cell cultures fail to capture how cells truly behave in complex 3D environments like the human body.

This limitation became especially problematic in tissue engineering, where scientists aim to build functional biological replacements using cells and supportive biomaterials called hydrogels. These water-rich, jelly-like substances mimic the body's natural scaffolding but present a formidable challenge: How do you see what's happening deep inside a living 3D structure without cutting it apart? Enter Optical Projection Tomography (OPT)—a breakthrough imaging technique turning hydrogels into transparent windows for cellular exploration 1 4 .

3D cell culture
Figure 1: 3D cell culture in hydrogel matrix (Credit: Unsplash)

What Makes OPT Different?

Principles of Light and Reconstruction

OPT operates on a deceptively simple principle: if light can pass through a sample, you can reconstruct its internal structure. Unlike confocal microscopy—which struggles beyond 0.3 mm depth—or two-photon microscopy (limited to ~0.5 mm), OPT handles samples up to 10 mm in diameter. Here's how it works:

1. Rotation & Projection

A hydrogel-encased sample is suspended in fluid and rotated while a camera captures hundreds of 2D images ("projections") from different angles.

2. Mathematical Magic

Using algorithms like filtered back projection (FBP), a 3D volume is reconstructed from these projections, similar to medical CT scans—but using visible light instead of X-rays 1 6 .

3. Label-Free or Fluorescent

OPT works in both transmission (brightfield) mode for label-free imaging and fluorescence mode to track specific molecules or live/dead cells 3 9 .

Why Hydrogels Love OPT

Hydrogels' secret lies in their optical clarity and refractive index, which closely matches water. This minimizes light scattering—a major hurdle for imaging opaque tissues. When cells are embedded inside, OPT visualizes their 3D distribution, morphology (shape), and density without toxic dyes or physical slicing. This non-destructive approach allows ongoing culturing, enabling time-lapse studies of cell behavior 1 7 .

Table 1: OPT vs. Competing Imaging Techniques
Technique Max Depth Resolution Sample Prep Live Imaging?
Confocal Microscopy 300 µm <1 µm Fluorescent labeling Limited
Two-Photon Microscopy 500 µm <1 µm Fluorescent labeling Yes
Micro-CT Unlimited ~10 µm Drying/coating No
OPT 10 mm 10-28 µm Minimal clearing Yes

Spotlight on Discovery: Decoding Cell-Hydrogel Interactions

The Central Experiment

A landmark 2021 study led by Belay et al. (Scientific Reports) tackled a critical question: How do different hydrogel formulations influence human cell behavior in 3D? To find out, they compared three hydrogels:

Gellan Gum (GG)

Basic, ionically crosslinked with spermidine.

Gelatin-Modified GG

Covalently bonded for enhanced cell attachment.

Geltrex

A commercial basement membrane matrix used as the "gold standard" 1 4 .

Methodology Step-by-Step

  • GG was chemically modified to create aldehyde-functionalized GG (GG-CHO).
  • Gelatin was treated with adipic dihydrazide (ADH) to generate hydrazide groups.
  • Mixing GG-CHO and gelatin-ADH formed hydrazone bonds, creating a covalently crosslinked network 1 .

  • Human fibroblasts were embedded in each hydrogel at 1 million cells/mL.
  • Samples were cast into 1.5-mm diameter cylinders—small enough for light penetration, large enough for physiological relevance.

  • Samples were immersed in sucrose solution (refractive index-matching) and rotated 360° in an OPT setup.
  • 400 projections per sample were captured using a 5x objective and sCMOS camera.
  • Reconstruction used FBP with corrections for illumination heterogeneity and center-of-rotation miscalibration 1 2 .

  • Cell Morphology: Cells were classified as "round," "spindle-like," or "spread" based on aspect ratios.
  • Cell Density: Voxel intensity in 3D reconstructions correlated to local cell density.
  • Viability: Fluorescent OPT distinguished live (green) vs. dead (red) cells.
Table 2: Key Findings from OPT Analysis
Hydrogel Type Cell Morphology (% elongated) Cell Density (cells/mm³) Viability (%) Notes
Gellan Gum (GG) 22% 8.7 × 10⁴ 85% Cells remained rounded
Gelatin-Modified GG 68% 1.2 × 10⁵ 92% Enhanced spreading
Geltrex® 75% 1.3 × 10⁵ 95% Highest cell activity

Why These Results Matter

The OPT data revealed a striking insight: Cells in gelatin-GG and Geltrex developed elongated, spindle-like shapes—indicating healthy attachment and interaction with their environment—while those in basic GG remained rounded and inactive. This quantitative link between material chemistry and cell response is vital for designing better tissue scaffolds. For instance, gelatin's integration into GG provided attachment sites mimicking natural collagen, explaining its superiority over plain GG 1 4 .

Microscopy image of cells
Figure 2: Cell morphology differences in various hydrogels (Credit: Unsplash)

The Scientist's Toolkit

OPT experiments rely on specialized materials and algorithms. Here's what powers this research:

Table 3: Essential Research Reagents & Tools
Item Function Example in Action
Gellan Gum (GG) Base polysaccharide for hydrogels; ionically crosslinkable Used in basic GG scaffolds 7
Spermidine (SPD) Ionic crosslinker for GG; forms bonds via polycation interactions Crosslinks GG in "GG 2% IO" hydrogels 2
Adipic Dihydrazide Modifies gelatin to form hydrazide groups for covalent crosslinking Creates stable gelatin-GG networks 1
Index-Matching Fluids Minimizes light refraction at sample boundaries (e.g., sucrose solution) Enables clear OPT imaging of hydrogels 1
FBP Algorithms Reconstructs 3D volumes from 2D projections Standard in OPT software 2 9
Variance Sharpness Correction Fixes rotational misalignment during reconstruction Critical for artifact-free images 2
Telecentric Lenses Ensures light rays parallel to optical axis, reducing distortion Used in OptiJ open-source OPT systems 8

Beyond the Basics: Where OPT is Heading

OPT is rapidly evolving through interdisciplinary innovations:

Resolution Revolution

Combining OPT with Gaussian Light Models (GBM) accounts for beam focusing effects, boosting resolution to sub-28 µm in platforms like those imaging mouse intestines 6 9 .

Open-Source Platforms

Projects like OptiJ—using 3D-printed parts and Fiji plugins—are democratizing access, slashing costs from ~$100,000 to under $5,000 8 .

Living Dynamics

Longitudinal tracking of stem cells in hydrogels now captures extracellular matrix production over weeks, revealing how tissues mature 3 .

Machine Learning

AI algorithms now segment cells 40% faster in OPT volumes, automating tasks like counting and morphology classification 1 .

Illuminating the Future

Optical Projection Tomography has transformed hydrogels from opaque blobs into dynamic, explorable ecosystems. By quantifying how cells navigate their 3D world—whether spreading, dividing, or secreting new tissue—OPT bridges materials science and biology. As one researcher poetically noted: "We're no longer just building scaffolds; we're choreographing cell symphonies in a glass theater." With faster, open, and higher-resolution OPT on the horizon, the next movement promises even deeper insights into regeneration, disease modeling, and the quest to print human tissues that truly live 1 6 8 .

For further reading, explore the original studies in Scientific Reports 1 4 or the open-source OptiJ project 8 .

References